This work was supported by an Australian Research Council Grant DP110100642 (to PT) and National Health and Medical Research Council Grants APP1002520 and APP1059426 (to PT and HYG).

NOTE: Toutes les expériences actuelles ont été réalisées conformément aux procédures locales d&#39;éthique animale de l&#39;Université de Queensland (approuvés par l&#39;Université du Queensland, Comité d&#39;éthique Biosciences anatomique). 1. Isolement îlots <ol><li> Préparation des réactifs <ol><li> Mélange d&#39;enzymes <ol><li> Pour la digestion du pancréas, utilisez un mélange de libérase et de la collagénase de type IV. Diluer libérase TL (thermolysine bas) 1 flacon de 5 mg avec 26 ml de DMEM (milieu de Eagle modifié Dulbecco). </li><li> Préparer la collagénase de type IV dans du tampon de Hank à une concentration de 0,5 mg / ml, supplémenté avec 5 mM de HEPES, 0,5 mM de CaCl2, 0,1 mg / ml de DNAse et de 1 mg / ml d&#39;albumine de sérum bovin. </li><li> Mélanger la TL Libérase et des solutions de collagénase dans un rapport de 4: 1 (en volume), soit 4 ml Libérase TL + 1 ml de collagénase. Aliquoter le mélange d&#39;enzymes pour 2,5 ml chacun et conserver à -20 ° C. NOTE: Les temps d&#39;incubation de tissu vvier un bit entre des lots d&#39;enzyme (30 sec à 1 min de différence). Par conséquent, un essai de traitement par lots peut être nécessaire. </li></ol></li><li> RPMI Isolation médias <ol><li> Dissoudre un flacon de poudre dans du milieu RPMI 1640 1 L d&#39;eau à la température ambiante et compléter avec 2 g de NaHCO 3, 4,02 g d&#39;HEPES. </li><li> Combiner les médias d&#39;isolement RPMI dans une grande fiole conique, mélanger avec un agitateur magnétique, ajuster à pH 7,4 avec NaOH, filtre stériliser et conserver à 4 ° C. </li></ol></li><li> RPMI Culture Media </li><li> Utilisation des milieux de culture RPMI avec 10% de sérum bovin fœtal et 1% d&#39;antibiotiques (par exemple, de la pénicilline 100 U / ml, streptomycine 100 pg / ml). </li></ol></li><li> Isolement et culture des Islet <ol><li> Sacrifice des animaux <ol><li> Sacrifiez la souris par dislocation cervicale ou CO 2 asphyxie selon les procédures d&#39;éthique de l&#39;institution locale. Vaporiser le corps de la souris à fond avec de l&#39;éthanol 70%. </li></ol></li><li> Perfusion pancréatique <ol><li> USE 2 coupes longues latéralement à travers la peau de l&#39;abdomen et du péritoine pour faire un rabat en forme de V. Tirez le rabat de la peau vers le haut pour découvrir la totalité de la cavité abdominale, mettre de côté l&#39;intestin et de garder le foie en place par un mouchoir en papier plié pour révéler le canal cholédoque (figures 1 et 2). Mettez l&#39;animal de sorte que la tête vers le chirurgien et la queue loin du chirurgien. </li><li> Appliquer une pince vasculaire à l&#39;ampoule sur la paroi du duodénum pour bloquer les enzymes injecté de pénétrer dans le duodénum (Figure 1). NOTE: Ceci est une étape importante car elle détermine si les enzymes injectées iront dans le canal pancréatique et perfuser le pancréas ou de reflux au site d&#39;injection (plus de pince) ou aller dans le duodénum (sous serre). </li><li> Cathétériser le canal cholédoque avec une aiguille 31 G près de la jonction du canal hépatique commun et canal cystique (Figure 1) et laisser la place à une deuxième injection si èmee pancréas ne parvient pas à perfuser la première fois. Lentement (~ 10 sec) injecter environ 2 ml de mélange d&#39;enzymes froid dans la souris canal cholédoque. NOTE: Le pancréas seront perfusés rapidement si la pince est dans la bonne position. </li><li> Ajustez la position de serrage si l&#39;aiguille est à l&#39;intérieur du canal cholédoque, mais ne peut pas perfuser le pancréas (ie., Se déplacer jusqu&#39;à la pince un peu loin du foie si plus serré ou vers le bas un peu pour le foie si, en vertu serré). REMARQUE: L&#39;aiguille va parfois dans la capsule qui entoure au lieu de la lumière du conduit. Un signe utile de vérifier si l&#39;aiguille est à l&#39;intérieur du canal cholédoque est la dilatation du canal lors de l&#39;injection. Si le pancréas ne parvient pas à perfuser, une alternative est le mélange d&#39;enzymes peut être injecté à plusieurs sites du pancréas avec un rendement plus faible prévu. La perfusion de l&#39;emplacement gauche du pancréas (le lobe splénique) est important pour la maximisation du rendement d&#39;îlots de Langerhans. </li><li> Après le pancréas est perfused, enlever rapidement le pancréas, le mettre dans un flacon (~ 20 ml, verre) et incuber pendant ~ 19 min dans un bain d&#39;eau à 37 C ° de. NOTE: Le temps d&#39;incubation peut varier un peu en fonction de la souche de souris et de l&#39;âge. </li><li> Après le temps d&#39;incubation, arrêter le processus de digestion en ajoutant ~ 20 ml de milieux d&#39;isolement froid. Agiter doucement le flacon pour perturber le pancréas et le verser dans un tamis de thé normal (diamètre de pore de 1 mm) dans un tube de 50 ml stérile. Compléter le tube avec les médias d&#39;isolement et centrifuger à 400 g pendant 1 min à 4 ° C avec frein off. </li><li> Dissoudre le culot dans les médias de l&#39;isolement et de transférer à nouveau à deux tubes de 15 ml, puis centrifuger à 400 g pendant 1 min à 4 ° C avec frein off. Après aspiration du surnageant, mélanger les deux pastilles bien avec 6 ml de milieu de séparation des cellules de gradient de densité (Histopaque 1077 solution) par tube au vortex ou aspiration et refoulement. Puis ajouter délicatement ~ 6 ml de milieux d&#39;isolement sur le côté du tube au-dessus deles milieux de séparation de gradient de densité et centrifuger à 100 g pendant 15 min à 4 ° C avec frein off. </li><li> Recueillir le surnageant dans le (milieux d&#39;isolement) supérieure, moyenne et inférieure (densité cellulaire gradient milieux de séparation) couche en 3 plats séparés. Retirez à la main les îlots dans ces trois plats, en utilisant une pipette sous un microscope à dissection. NOTE: La couche intermédiaire contient la plupart des îlots. Les îlots sont reconnus comme des structures compactes de ~ 50 à 500 um de taille (Figure 3). Nous obtenons ~ 200 îlots de souris WT et 300 à &lt;100 îlots de souris db / db. </li></ol></li><li> La culture Islet <ol><li> Pour aider les îlots se remettre de la digestion enzymatique, des groupes de culture de 40 îlots dans une boîte de Pétri de 25 cm avec 6 ml RPMI milieux de culture (10 mM de glucose) à 37 ° C, 5% / 95% de CO 2 / O 2 pendant 2 jours avant en direct -cell 2 photons imagerie. Changer les milieux de culture quotidienne. </li></ol></li></ol></li></ol> 2. Live Cell 2-Photon Imaging <ol><li> Préparation des réactifs <ol><li> Extracellulaire Buffer <ol><li> Préparer le tampon de sodium extracellulaire riche pour le dosage de deux photons avec 140 mM de NaCl, 5 mM de KCl, 2,5 mM de CaCl2, 1 mM de MgCl2, 5 mM de NaHCO 3 et 10 mM de HEPES. </li></ol></li><li> Extracellulaire Dye <ol><li> Pour le test 2-photon, préparer stocks SRB 8 mM dans un tampon extracellulaire, aliquote dans des petits tubes et conserver à -20 ° C. Diluer le stock SRB 1:10 dans un tampon extracellulaire frais pour obtenir une concentration de travail de 0,8 mm. </li></ol></li></ol></li><li> photon Imaging <ol><li> Tout d&#39;abord, pré-incubation de 2 jours îlots cultivés en 3 mM de tampon glucose extracellulaire pendant 30 min. NOTE: Nous utilisons îlots entre 2 et 5 jours dans la culture. </li><li> Ensuite, placez une lame de verre sur la chambre thermo-commande monté sur la platine du microscope. Utiliser de la graisse pour empêcher la fuite de solution. </li><li> Couvrir la chambre avec 500 pi de extracellparti- tampon contenant 0,8 mM SRB avec des concentrations de glucose différentes (3 mm, 6 mm, 15 mm et 20 mM de glucose). </li><li> Ensuite, placer l&#39;îlot pré-incubées dans le milieu de la chambre et de se concentrer à 3-4 couches de cellules dans l&#39;îlot où, dans les îlots de rongeurs, immunocoloration pour l&#39;insuline montre que la plupart des cellules sont des cellules bêta. NOTE: Le SRB décrira tous la membrane cellulaire de l&#39;îlot rapidement et l&#39;îlot est prêt à l&#39;image. </li><li> Utilisation d&#39;un microscope à 2 photons avec un objectif 60X à immersion dans l&#39;huile (1,42 NA) et un logiciel d&#39;imagerie (par exemple., ScanImage 11). La figure 4 illustre un dispositif de microscopie classique. Image des événements d&#39;exocytose utilisant SRB comme une membrane impermeant marqueur extracellulaire fluorescente excitée par des impulsions laser femtosecondes à 880 nm, avec fluorescence détectée à 550-650 nm. Notez les réponses des îlots à la stimulation. </li><li> Identifier les événements d&#39;exocytose simples par l&#39;apparition soudaine d&#39;une petite tache fluorescente (résolution de 10 pixels /1; m) et confirmer par la phase de montée rapide du signal fluorescent sur ​​une région d&#39;intérêt (~ 0,78 um 2). </li></ol></li></ol>

<ol>
	<li>Guariguata, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+estimates+of+diabetes+prevalence+for+2013+and+projections+for+2035.">Global estimates of diabetes prevalence for 2013 and projections for 2035.</a> Diabetes Research and Clinical Practice. 103, 137-149 (2014).</li><li>Ashcroft, F. M., Rorsman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+mellitus+and+the+beta+cell:+The+last+ten+years.">Diabetes mellitus and the beta cell: The last ten years.</a> Cell. 148, 1160-1171 (2012).</li><li>Weir, G. C., Bonner-Weir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Five+stages+of+evolving+beta-cell+dysfunction+during+progression+to+diabetes.">Five stages of evolving beta-cell dysfunction during progression to diabetes.</a> Diabetes. 53, S16-S21 (2004).</li><li>Kjorholt, C., Akerfeldt, M. C., Biden, T. J., Laybutt, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+hyperglycemia,+independent+of+plasma+lipid+levels,+is+sufficient+for+the+loss+of+beta-cell+differentiation+and+secretory+function+in+the+db/db+mouse+model+of+diabetes.">Chronic hyperglycemia, independent of plasma lipid levels, is sufficient for the loss of beta-cell differentiation and secretory function in the db/db mouse model of diabetes.</a> Diabetes. 54, 2755-2763 (2005).</li><li>Wang, Y. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+type+2+diabetic+rodent+models.">Spontaneous type 2 diabetic rodent models.</a> Journal of Diabetes Research. , (2013).</li><li>Hummel, K. P., Dickie, M. M., Coleman, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+a+new+mutation+in+mouse.">Diabetes a new mutation in mouse.</a> Science. 153, 1127-1128 (1966).</li><li>Do, O. H., Low, J. T., Gaisano, H. Y., Thorn, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secretory+deficit+in+islets+from+db/db+mice+is+mainly+due+to+a+loss+of+responding+beta+cells.">The secretory deficit in islets from db/db mice is mainly due to a loss of responding beta cells.</a> Diabetologia. 57, 1400-1409 (2014).</li><li>Low, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+principally+regulates+insulin+secretion+in+mouse+islets+by+controlling+the+numbers+of+granule+fusion+events+per+cell.">Glucose principally regulates insulin secretion in mouse islets by controlling the numbers of granule fusion events per cell.</a> Diabetologia. 56, 2629-2637 (2013).</li><li>Takahashi, N., Kishimoto, T., Nemoto, T., Kadowaki, T., Kasai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+pore+dynamics+and+insulin+granule+exocytosis+in+the+pancreatic+islet.">Fusion pore dynamics and insulin granule exocytosis in the pancreatic islet.</a> Science. 297, 1349-1352 (2002).</li><li>Low, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+secretion+from+beta+cells+in+intact+mouse+islets+is+targeted+towards+the+vasculature.">Insulin secretion from beta cells in intact mouse islets is targeted towards the vasculature.</a> Diabetologia. 57, 1655-1663 (2014).</li><li>Pologruto, T. A., Sabatini, B. L., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScanImage:+flexible+software+for+operating+laser+scanning+microscopes.">ScanImage: flexible software for operating laser scanning microscopes.</a> Biomedical Engineering Online. 2, 13 (2003).</li></ol>

The authors declare they have no competing financial interests.

Le facteur le plus critique dans l&#39;isolement d&#39;îlots est la perfusion initiale du pancréas; un sous perfusion du pancréas résultats avec un rendement d&#39;îlots considérablement plus faible. D&#39;autres facteurs affectent également la qualité de l&#39;isolation tel que le temps de digestion et le niveau d&#39;agitation qui peut compenser en partie au niveau de la perfusion. Par exemple, un pancréas perfusés pleinement devra être incubées à 37 ° C pendant ~ 18 min 30 sec tout en agitant doucement,...

Le rôle d&#39;un dysfonctionnement des cellules bêta dans la maladie a été largement reconnu 3,4. Des lignées cellulaires telles que la MIN6 et INS-1 sont des outils utiles pour comprendre la biologie de comportement des cellules bêta. Toutefois, le contrôle physiologique de la sécrétion d&#39;insuline a lieu dans les îlots de Langerhans. Ces îlots contiennent des milliers de cellules bêta serrées, ainsi que les vaisseaux sanguins et d&#39;autres types de cellules endocrines. Cet environnement au sein de l&#39;îlot influe sur la sécrétion d&#39;insuline et est susceptible d&#39;être important dans le diabète. Par conséquent, pour comprendre le contrôle physiologique de la sécrétion d&#39;insuline, et la physiopathologie de la maladie, il est essentiel d&#39;étudier îlots intacts. isolement des îlots Îlots humains vivants et, en particulier, des îlots humains provenant de patients atteints de diabète de type 2 sont difficiles à obtenir. En outre, des îlots humains ont des possibilités limitées pour la manipulation moléculaire expérimentale. Les chercheurs ont donc utilisé estpermet à partir d&#39;animaux et des modèles animaux de diabète de type 2. Un tel modèle de la maladie est la souris db / db. Ceci est une mutation spontanée que les modèles diabète de type 2 avec une progression de phénotype qui se rapproche étroitement de la maladie humaine 5,6. Le protocole présenté ici pour isolement des îlots de souris db / db diabétiques comporte de nombreuses étapes en commun avec d&#39;autres groupes avec quelques améliorations pour un meilleur rendement, la purification et le renforcement de la survie de l&#39;îlot. 2 imagerie photonique Le dosage des cellules vivantes 2 photons décrit ici permet aux chercheurs de quantifier le nombre et évaluer les caractéristiques des granulés contenant de l&#39;insuline à partir de cellules simples de 7 et diabétique de type sauvage îlots 8,9.

<table border="0" cellpadding="0" cellspacing="0" width="675">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:173px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Liberase TL 5mg</td>
			<td style="width:173px;">Roche</td>
			<td style="width:119px;">-5401020001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase type IV-1g</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">17104-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Histopaque 1077 500ml</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:119px;">10771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 Medium (10X) 1L</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:119px;">R1383</td>
			<td>to prepare isolation media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 (1X) 500ml</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">21870-076</td>
			<td>to prepare cultured media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin-Streptomycin 100ml</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td>to prepare cultured media&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal bovine serum 500ml</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">10099-141</td>
			<td>to prepare cultured media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM (Dulbecco&#39;s Modified Eagle Medium)</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">11966-025</td>
			<td>to dilute the liberase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Metamorph program</td>
			<td style="width:173px;">Molecular Devices, USA</td>
			<td style="width:119px;"></td>
			<td>to analyze the 2-photon images</td>
		</tr>
	</tbody>
</table>

leprdb mouse model of type 2 diabetes: pancreatic islet isolation and live-cell 2-photon imaging of intact islets

Le diabète de type 2 est une maladie chronique qui touche 382 millions de personnes en 2013, et devrait atteindre 592 millions d&#39;ici 2035 1. Au cours des deux dernières décennies, le rôle de la dysfonction des cellules bêta dans le diabète de type 2 a été clairement établi 2. progrès de la recherche a nécessité des procédés pour l&#39;isolement des îlots pancréatiques. Le protocole de l&#39;isolement des îlots présenté ici part de nombreuses étapes communes avec des protocoles d&#39;autres groupes, avec quelques modifications pour améliorer le rendement et la qualité des îlots isolés à la fois du type sauvage et db Lepr diabétique de souris (db / db). Un des cellules vivantes Procédé d&#39;imagerie à 2 photons est ensuite présenté qui peut être utilisée pour étudier le contrôle de la sécrétion d&#39;insuline des îlots à l&#39;intérieur.

Rendement Islet et la purification Pour un type sauvage souris normale, environ 200 îlots sont attendus. Îlots sains regarder lumineux, de forme ronde et avoir une frontière lisse. Un lot d&#39;isolement sur ​​digéré a généralement petites et floues îlots tandis qu&#39;un lot de sous-digéré a moins îlots et attachée cellules îlots acineuses (Figure 3). Pour les souris diabétiques db / db, le rendement de l&#39;îlot et l&#39;apparence dépend de la progress...

Watch this Scientific Journal Video about Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets at JoVE.com

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Lepr<sup&gt; Db</sup&gt; Modèle de souris de diabète de type 2: pancréatique isolement des îlots et des cellules vivantes 2-Photon Imaging d&#39;îlots intacts

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

leprdb-mouse-model-type-2-diabetes-pancreatic-islet-isolation-live

Research

JoVE Journal

Medicine

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

9.3K vues.  The University of Queensland. We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Vidéo: Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

L&#39;isolement des îlots humains à partir des patients partiellement pancréatectomisés

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Recherche

Médecine

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Protocoles de coloration pour des îlots pancréatiques humains

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes.
Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression1. They lack chronic toxicity because they lack viral coding sequences2-5 and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes.
In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice.

We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression.

Neo-Islet formation dans le foie des souris diabétiques par Helper-dépendante Transfert de gènes à médiation adénovirale Vecteur

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the ...

Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

Kidney diseases involving urinary loss of large essential macromolecules, such as serum albumin, have long been thought to be caused by alterations in the permeability barrier comprised of podocytes, vascular endothelial cells, and a basement membrane working in unison. Data from our laboratory using intravital 2-photon microscopy revealed a more permeable glomerular filtration barrier (GFB) than previously thought under physiologic conditions, with retrieval of filtered albumin occurring in an early subset of cells called proximal tubule cells (PTC)1,2,3.
Previous techniques used to study renal filtration and establishing the characteristic of the filtration barrier involved micropuncture of the lumen of these early tubular segments with sampling of the fluid content and analysis4. These studies determined albumin concentration in the luminal fluid to be virtually non-existent; corresponding closely to what is normally detected in the urine. However, characterization of dextran polymers with defined sizes by this technique revealed those of a size similar to serum albumin had higher levels in the tubular lumen and urine; suggesting increased permeability5.
Herein is a detailed outline of the technique used to directly visualize and quantify glomerular fluorescent albumin permeability in vivo. This method allows for detection of filtered albumin across the filtration barrier into Bowman's space (the initial chamber of urinary filtration); and also allows quantification of albumin reabsorption by proximal tubules and visualization of subsequent albumin transcytosis6. The absence of fluorescent albumin along later tubular segments en route to the bladder highlights the efficiency of the retrieval pathway in the earlier proximal tubule segments. Moreover, when this technique was applied to determine permeability of dextrans having a similar size to albumin virtually identical permeability values were reported2. These observations directly support the need to expand the focus of many proteinuric renal diseases to included alterations in proximal tubule cell reclamation.

A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states.

Quantifier glomérulaire perméabilité des macromolécules fluorescentes en microscopie 2 photons à Munich chez le rat Wistar

Kidney  diseases involving urinary loss of large essential macromolecules, such as  serum albumin, have long been thought to be caused by alterations in ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

L&#39;isolement, la culture, et de l&#39;imagerie de l&#39;homme causés par des amas de cellules pancréatiques

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

Contraste dynamique amélioré imagerie par résonance magnétique d&#39;un modèle orthotopique cancer du pancréas de souris

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide new expandable sources of beta cells to alleviate the donor scarcity in human islet transplantation as therapy for diabetes. Although recent advances have been made towards this aim, mechanisms that regulate beta cell expansion and differentiation from a stem/progenitor cell remain to be characterized. Here, we describe a protocol for an injury model in the adult mouse pancreas that can function as a tool to study mechanisms of tissue remodeling and beta cell proliferation and differentiation. Partial duct ligation (PDL) is an experimentally induced injury of the rodent pancreas involving surgical ligation of the main pancreatic duct resulting in an obstruction of drainage of exocrine products out of the tail region of the pancreas. The inflicted damage induces acinar atrophy, immune cell infiltration and severe tissue remodeling. We have previously reported the activation of Neurogenin (Ngn) 3 expressing endogenous progenitor-like cells and an increase in beta cell proliferation after PDL. Therefore, PDL provides a basis to study signals involved in beta cell dynamics and the properties of an endocrine progenitor in adult pancreas. Since, it still remains largely unclear, which factors and pathways contribute to beta cell neogenesis and proliferation in PDL, a standardized protocol for PDL will allow for comparison across laboratories.

This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation.

Blessure chirurgicale au pancréas de souris par ligature du canal pancréatique tant que modèle de endocrines et exocrines Reprogrammation et prolifération

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide ...

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Intraporte Transplantation d’îlots pancréatiques dans le modèle murin

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Mesure de la contractilité isométrique de l’artère mésentérique de souris à l’aide de fil myographie

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology have been developed and utilized in vivo, they are limited in terms of allowing single cell resolution and continuous longitudinal recordings. Because of the transparency of the cornea, the anterior chamber of the eye (ACE) in mice is well suited to study human and mouse pancreatic islet cell biology. Here is a description of how this approach can be used to perform continuous longitudinal recordings of grafting and revascularization of individual human islet grafts. Human islet grafts are inserted into the ACE, using NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mice as recipients. This allows for the investigation of the expansion of recipient versus donor cells and the contribution of recipient cells in promoting the encapsulation and vascularization of the graft. Further, a step-by-step approach for image analysis and quantification of the islet volume or segmented vasculature and islet capsule forming recipient cells is outlined.

The goal of this protocol is to continuously monitor the dynamics of the human pancreatic islet engraftment process and the contributing host versus donor cells. This is accomplished by transplanting human islets into the anterior chamber of the eye (ACE) of an NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mouse recipient followed by repeated 2-photon imaging.

Imagerie in vivo longitudinale et quantification des îlots pancréatiques humains Greffant et contribution des cellules hôtes dans la chambre oculaire antérieure

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology ...